Ablation of Nonmuscle myosin II-C and Double Ablation of NM II-B and II-C in Mice: Ablation of nonmuscle myosin (NM) II-A or NM II-B results in mouse embryonic lethality. In this project we ablated NM II-C as well as NM II-C/II-B together in mice. NM II-C ablated mice survive to adulthood and show no obvious defects compared to wild-type littermates. However, ablation of NM II-C in mice expressing only 12% of wild-type amounts of NM II-B results in a marked increase in cardiac myocyte hypertrophy compared to the NM II-B hypomorphic mice alone. In addition, these hearts develop interstitial fibrosis associated with diffuse N-cadherin and beta-catenin localization at the intercalated discs, where both NM II-B and II-C are normally concentrated. When both NM II-C and II-B are ablated the B-C-/B-C- cardiac myocytes show major defects in karyokinesis. Over 90% of B-C-/B-C- myocytes demonstrate defects in chromatid segregation and mitotic spindle formation accompanied by increased stability of microtubules and abnormal formation of multiple centrosomes. This requirement for NM II in karyokinesis is further demonstrated in the HL-1 cell line derived from mouse atrial myocytes, using siRNA knockdown of NM II or treatment with the myosin inhibitor blebbistatin. Our study shows that NM II is involved in regulating cardiac myocyte karyokinesis by affecting microtubule dynamics. Cardiac Specific Ablation of Nonmuscle Myosin II-B in Mice: A Role of Nonmuscle Myosin II-B in Coronary Vessel Formation during Mouse Heart Development Mice ablated for nonmuscle myosin II-B (NM II-B) die by E14.5 due to heart defects. To better understand the role of NM II-B in late heart development we used transgenic mice expressing Cre-recombinase driven by the SM22alpha promoter to conditionally ablate NM II-B in cardiac myocytes and epicardial cells but not in endocardial and fibroblast cells. BSM22alpha/BSM22alpha mice developed arrhythmias as early as 1 month old and died suddenly by 6 months in dilated right ventricular failure. Analysis of BSM22alpha/BSM22alpha hearts shows significant defects in coronary vessel formation especially in the right ventricle with progressive loss of right ventricular cardiac myocytes. BSM22alpha/BSM22alpha hearts are also hypoplastic with defects in cytokinesis in the cardiac myocytes. To further study the cellular mechanisms of coronary vessel defects in BSM22alpha/BSM22alpha hearts, we ablated NM II-B specifically in epicardial (BWT-1/BWT-1 mice) or myocardial (BNkx/BNkx mice) cells using WT-1 and Nkx2.5 Cre mice, respectively. Both BWT-1/BWT-1 and BNkx/BNkx mice died during embryonic development with hypoplastic hearts. Interestingly, while the BWT-1/BWT-1 hearts showed severe defects in coronary vessel formation, BNkx/BNkx hearts showed marked defects in cardiac myocyte cytokinesis. Consistent with abnormal coronary vessel formation in BWT-1/BWT-1 hearts, studies employing epicardial explants in collagen gels show that NM II-B ablated epicardial cells develop defects in branching morphogenesis. Similar defects were observed in wild type epicardial explants when treated with blebbistatin which inhibits NM II enzymatic activity. In addition epicardial explants prepared from mice expressing motor impaired NM II-B also show branching abnormalities, indicating a requirement for NM II-B enzymatic (motor) activity. Our results provide evidence for a novel role for NM II-B in coronary vessel formation during mouse heart development. Conditional Ablation of Nonmuscle Myosin II-B: Cre-recombinase Controlled by the Nestin or alpha-cardiac Myosin Heavy Chain Promotor We used a loxP/Cre recombinase strategy to specifically ablate nonmuscle myosin II-B (NM II-B) in the brains or hearts of mice. These are the two major organs affected by germ line ablation of NM II-B (B-/B- mice). Our purpose was two-fold: first to identify the cell-type(s) responsible for the defects found in the hearts and brains of B-/B- mice and second, to avoid the embryonic lethality found in B-/B- mice. Mice ablated for NM II-B in neural tissues (Bnest/Bnest mice) develop severe hydrocephalus starting at E16.5 and die between postnatal day (P)12 and 22 without showing cardiac defects. Mice deficient in NM II-B only in cardiac myocytes (BalphaMHC/BalphaMHC mice) do not show defects in the brain. However BalphaMHC/BalphaMHC mice display novel cardiac defects not seen in B-/B- mice. Between 6-10 months most BalphaMHC/BalphaMHC mice develop a dilated cardiomyopathy, which includes interstitial fibrosis and infiltration of the myocardium and pericardium with inflammatory cells. At 10 months echocardiography measures a decrease in fractional shortening of BalphaMHC/BalphaMHC hearts. In addition most of the BalphaMHC/BalphaMHC mice showed an abnormal right axis by EKG. Four of five BalphaMHC/BalphaMHC hearts develop marked widening of the intercalated discs, consistent with a role for NM II-B in myocyte cell adhesion. By avoiding the embryonic lethality found in germline-ablated mice we were able to study the role of NM II-B in adult mice and to define a role for this isoform in the intercalated disc.